This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Since the late 80s nitric oxide has been recognized to have many important physiological roles. For example NO has important signaling functions and is a potent antimicrobial agent active against a wide range of bacteria viruses fungi and even parasites. Nitric oxides physiological functions almost invariably involve specific interactions with proteins;much progress has been made in identifying the mechanisms of such protein-NO interactions but many questions remain. Herein we propose to develop broadly applicable methodologies for photochemically generating free NO within a protein crystal so that subsequent interactions of NO with the protein can be monitored in real time using time-resolved protein X-ray crystallography. Such methodologies will be invaluable tools for studying the detailed mechanisms of NO signaling and antimicrobial phenomena. The project will be divided into 2 phases. In phase 1 various methods for NO photogeneration will be tested on crystals of bovine liver catalase and superoxide dismutase (SOD) from Propionibacterium shermanii. Both are readily available proteins whose crystal structures are known and that interact strongly with NO. In the second phase of the project the best NO photogenerating systems will be used to investigate NO interaction with oxygenated HbN from M. tuberculosis. HbN is a protein that is thought to protect M. tuberculosis from the bodys defenses and allow TB to persist for years in a latent state. The aim of this phase will be to show that our method can be applied to a system of considerable importance in medicine and public health.